The production of chemicals in bioreactor systems is expensive. The differential between production costs and product market value is the dominant driving force for drug discovery and development of potential bioprocesses. The importance of production costs is reflected in the economic observation that the volumetric productivity of a wide range of biologically produced products is about the same at $0.17 per liter per day. The expense of biological production is a motivation for pursuing chemical synthesis when possible; however, the complexity of synthesis of many natural products often makes this route equally costly. In the absence of chemical synthesis, metabolites derived from microorganisms must be produced in aseptic bioreactors. In contrast, plant-derived chemicals can be harvested from intact plants. Therefore, agronomic production or collection from natural environments is a formidable competitor to growth of plant tissues in bioreactor systems. Many rationales are given for pursuing plant tissue culture as a potential production system. The most compelling are those situations where intact plants are poor competitors. Some plants either grow very slowly, or are not amenable to agronomic production. In addition, environmental degradation is limiting the attractiveness of natural harvest, particularly from endangered environments such as the rain forest where the biochemical diversity is the greatest.
Although the bioreactor described herein is not limited to use for plant tissue culture, the economic constraints and stringent asepsis requirements presented by this production system provide an excellent context to demonstrate the effectiveness of the bioreactor. There have been many efforts to commercialize plant metabolites from cell culture; however, few have achieved commercial success. Low productivity is usually cited as the reason for failure despite the fact that production rates and tissue concentrations are very often substantially higher than the intact plant. In fact, tremendous productivities have been achieved by plant tissue culture. There are at least eight different systems where the metabolite levels are greater than 10% of the cell dry weight, and several of these productivities have been achieved with cultures that display relatively high growth rates. One example is anthocyanin pigments production by P.C.C. Technology, Japan where the cell content was greater than 17% and effective specific growth rates were 0.22 day.sup.-1 at a scale of 500 L. Rosmarinic acid production was successfully scaled up by A Nattermann & Cie GmbH in a 30 L stirred tank. The titer of rosmarinic acid reached 5.5 g/L with volumetric productivity of nearly 1 g/L/day, and tissue content as high as 21% of dry weight. The failure of these processes is more a failure to compete economically with whole plant material rather than a failure of the cultures to be biochemically productive. There have also been significant advances in strategies to improve cellular productivity by cell line selection, genetic engineering, elicitation and root culture or enhance reactor productivity by operational strategies such as high density culture, integrated product recovery and immobilization. However, there is a limit to the improvements that can be achieved by these strategies, and for compounds where there is a low-cost alternative from intact plant material, it simply does not make sense to attempt production in bioreactor systems.
Despite the limitations, the potential of plant-tissue culture derived chemicals has resulted in a tremendous investment from both industry and academia in developing this technology. It has been demonstrated that large-scale production is technically feasible. The first commercial process was the production of shikonin. Since the market for this dye is limited, production has been on hold to focus efforts on taxol as a more profitable target. Similarly, the efforts of EscaGenetics on the production of vanillin were way-layed in favor of taxol development. Ginseng has been produced commercially by Nitto Denko (Japan) for 10 years at a scale of 25,000 liters. There are other reports of industrial scale cultivation of plant cells including tobacco at 15,500 L and three different plant species by DIVERSA (Hamburg, Germany) up to 75,000 L. Taxus sp. is being grown at industrial scale by both Phyton, Inc. and Sam Yang. These examples show that technical problems of scale-up can be overcome.
The preceding indicates that the technology for production of chemicals by plant tissue culture is available provided the secondary metabolite has a sufficiently high value. The required product price to consider plant tissue culture production has been estimated to be in the range of $1000 to $5000 per Kg. The issue arises as to whether this technology can be extended to lower value/higher volume biochemical production. To achieve this objective, it is useful to understand what contributes to production costs.
Based on experience with the commercial development of shikonin, the Mitsui group estimated that 64% of the production costs for cultured plant cells was due to fixed costs (depreciation, interest and capital expenditures). A similar number can be calculated from the recent analysis presented by Goldstein based on general plant tissue culture characteristics. Using Goldstein's 2000 kg product per year basis (which is implicitly 22 tons of cell mass based on assumed productivity), the fixed costs (calculated as capital charges) were 55.4% of the manufacturing costs. The estimate of Yoshioka and Fujita is likely to be more generally applicable since it uses a cycle time of roughly 14 days as compared to the 5-day reactor cycle time assumed by Goldstein. Clearly capital investment is an important target for cost reduction. This is not surprising since equipment and support facilities associated with aseptic bioprocessing are extremely expensive because vessels are constructed of stainless steel and pressure rated for autoclave sterilization. Accordingly, eliminating the need for expensive autoclave construction could substantially reduce production costs by reducing the initial capital investment.